The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H2O2 in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H2O2 are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H2O2 and is sensitive enough to assay mitochondrial H2O2 generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H2O2. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.